INFECTION AND IMMUNITY, Oct. 2004, p. 5630–5637 Vol. 72, No.

10
0019-9567/04/$08.00⫹0 DOI: 10.1128/IAI.72.10.5630–5637.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Serum Levels of the Proinflammatory Cytokines Interleukin-1 Beta

(IL-1␤), IL-6, IL-8, IL-10, Tumor Necrosis Factor Alpha, and
IL-12(p70) in Malian Children with Severe Plasmodium
falciparum Malaria and Matched Uncomplicated
Malaria or Healthy Controls
K. E. Lyke,1 R. Burges,1 Y. Cissoko,2 L. Sangare,2 M. Dao,2 I. Diarra,2 A. Kone,2
R. Harley,1 C. V. Plowe,1 O. K. Doumbo,2 and M. B. Sztein1*
Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland,1 and Department

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of Epidemiology of Parasitic Diseases, Malaria Research and Training Center, Bandiagara
Malaria Project, University of Bamako, Bamako, Mali2
Received 9 September 2003/Returned for modification 22 December 2003/Accepted 4 July 2004

Inflammatory cytokines play an important role in human immune responses to malarial disease. However,
the role of these mediators in disease pathogenesis, and the relationship between host protection and injury
remains unclear. A total of 248 cases of severe Plasmodium falciparum malaria among children aged 3 months
to 14 years residing in Bandiagara, Mali, were matched to cases of uncomplicated malaria and healthy
controls. Using modified World Health Organization criteria for defining severe malaria, we identified 100
cases of cerebral malaria (coma, seizure, and obtundation), 17 cases of severe anemia (hemoglobin, <5 g/dl),
18 cases combined cerebral malaria with severe anemia, and 92 cases with hyperparasitemia (asexual tropho-
zoites, >500,000/mm3). Significantly elevated levels (given as geometric mean concentrations in picograms/
milliliter) of interleukin-6 (IL-6; 485.2 versus 54.1; P ⴝ <0.001), IL-10 (1,099.3 versus 14.1; P ⴝ <0.001),
tumor necrosis factor alpha (10.1 versus 7.7; P ⴝ <0.001), and IL-12(p70) (48.9 versus 31.3; P ⴝ 0.004) in
serum were found in severe cases versus healthy controls. Significantly elevated levels of IL-6 (485.2 versus
141.0; P ⴝ <0.001) and IL-10 (1,099.3 versus 133.9; P ⴝ <0.001) were seen in severe malaria cases versus
uncomplicated malaria controls. Cerebral malaria was associated with significantly elevated levels of IL-6
(754.5 versus 311.4; P ⴝ <0.001) and IL-10 (1,405.6 versus 868.6; P ⴝ 0.006) compared to severe malaria cases
without cerebral manifestations. Conversely, lower levels of IL-6 (199.2 versus 487.6; P ⴝ 0.03) and IL-10
(391.1 versus 1,160.9; P ⴝ 0.002) were noted in children with severe anemia compared to severe malaria cases
with hemoglobin at >5 g/dl. Hyperparasitemia was associated with significantly lower levels of IL-6 (336.6
versus 602.1; P ⴝ 0.002). These results illustrate the complex relationships between inflammatory cytokines
and disease in P. falciparum malaria.

Inflammatory cytokines play an important role in human
immune responses to malaria disease, although the balance
between pro- and anti-inflammatory cytokines and the patho-
genic effects that can result from dysregulation are poorly
understood. Malaria disease manifestations differ and appear
to be regulated by age and the acquisition of immunity, host
and parasite genetic polymorphisms, and regional variation.
Variations in human cytokine responses and their link to ma-
laria disease manifestations are the subject of much debate.
Differential activation of CD4⫹-T-cell populations and re-
sultant cytokine activity in malaria has been well defined in
murine models. Cytokine production (tumor necrosis factor
alpha [TNF-␣] and gamma interferon [IFN-␥]) appears neces-
sary for the inhibition of parasitemia (6, 11, 25, 43) and stim-
ulation of phagocytosis to enhance clearance of parasitized
erythrocytes (35). Intraperitoneal injection of mice with re-
combinant interleukin-12 (IL-12) appears to induce IFN-␥-
* Corresponding author. Mailing address: The University of Mary-
land at Baltimore, Center for Vaccine Development, 685 W. Baltimore
St., HSF 480, Baltimore, MD 21201. Phone: (410) 706-2345. Fax: (410)
706-6205. E-mail: msztein@medicine.umaryland.edu.
mediated protection against Plasmodium yoelii sporozoite chal-
lenge, and IL-12 injection before sporozoite challenge was
found to protect monkeys against malaria (23, 44). Paradoxi-
cally, these cytokines are also implicated in the pathology of
complicated malaria. T-cell-deficient nude mice do not de-
velop cerebral manifestations upon parasite challenge (12). In
addition, anti-TNF-␣ and anti-IFN-␥ antibodies appear able to
abolish the onset of cerebral malaria (17, 19). Of note, IL-10
has been proposed to downregulate Th1 cytokine production,
resulting in a lower incidence of cerebral symptoms in mice
coinfected with Plasmodium berghei and IL-10-stimulating LP-
BM5 murine leukemia virus (10).
In humans, the role of inflammatory cytokines in malaria is
less well defined. Levels of endogenous pyrogens, such as IL-6,
IL-1␤, and IL-8, are elevated in malaria disease (13, 34) and
correlate with disease severity (8, 26, 28, 48, 50). TNF-␣ ap-
pears to be pivotal both in the early response to malaria and in
late, pathological manifestations. Excess production of TNF-␣
is likely to be involved in the appearance of symptoms such as
fever and headache associated with malaria disease (5, 30), and
it has been linked to disease severity and complications (19, 28,
45). The mechanism may relate, in part, to increases in eryth-

5630
VOL. 72, 2004 INFLAMMATORY CYTOKINES IN P. FALCIPARUM MALARIA
rocyte cytoadherence induced by proinflammatory cytokines
via upregulation of adhesion molecules on vascular endothe-
lium (22). Reverse transcriptase-PCR postmortem analysis of
human brain tissue in patients who succumbed to cerebral
malaria demonstrates expression of TNF-␣ and IL-1␤ (2, 41,
47). However, in contrast to observations in the murine model,
monoclonal antibodies to TNF-␣ have been shown in humans
to ameliorate fever but not the manifestations of cerebral ma-
laria (29).
It is widely accepted that Th2 cytokines downregulate Th1-
derived cytokines. Elevated levels of anti-inflammatory IL-10
have been reported in severe malaria (40, 42). Murine and in
vitro studies have demonstrated IL-10’s ability to inhibit
TNF-␣ production in response to malarial antigens (21).
Moreover, IL-10 has been shown in other models to prevent
TNF-␣-associated lethal endotoxemia (14, 24) and inhibit an-
tigen-induced lymphoproliferation by downregulating major
histocompatibility complex class II antigen expression on
monocytes (9). Thus, IL-10 appears to play an important role
in counteracting the potentially harmful host proinflammatory
response to malaria antigens.
Most of the studies described above provide only fragmen-
tary information in relatively small numbers of volunteers,
precluding a comprehensive evaluation of the role of cytokines
in the pathogenesis of the various malaria disease manifesta-
tions. Thus, in the present study we sought to further under-
stand the complexities of cytokine responses and their role in
the pathogenesis of clinical malaria by correlating cytokine
responses to clinical disease, with an emphasis on patients with
severe-malaria manifestations. To this end, we comprehen-
sively examined the patterns of inflammatory cytokines [IL-1␤,
IL-6, IL-8, IL-10, IL-12(p70), and TNF-␣] in serum by using a
matched case-control format, involving a large population of
African children with manifestations of severe malaria and
uncomplicated malaria, as well as healthy controls.
MATERIALS AND METHODS
Study site and enrollment. Serum was obtained from 776 Malian children
(aged 3 months to 14 years) on enrollment into a case-control study evaluating
risk and protective factors for severe malaria. The study site of Bandiagara
(population, 13,600) is located in east central Mali in West Africa and has intense
seasonal transmission (July to December) of P. falciparum malaria. The predom-
inant ethnic group in the study area is Dogon (65%), with Peuhl (10%), Bambara
(15%), and other ethnic groups (10%) also represented.
A total of 258 index cases of severe malaria from Bandiagara and surrounding
areas were admitted to the Bandiagara Malaria Project ward from October 1999
to January 2003. Cases were classified as severe malaria based on modified
criteria put forth by the World Health Organization (51) (Table 1). More than
one clinical diagnosis for severe malaria was possible, but cerebral malaria and
severe anemia were considered to be the primary defining features when they
coexisted with other criteria. Each index case was age, residence, and ethnicity
matched to a case of uncomplicated malaria and a healthy control.
The following definitions were used to match controls to the index case. Age
categories were defined as 3 to 5 months, 6 to 11 months, 1 year, 2 years, 3 to 4
years, 5 to 6 years, 7 to 8 years, 9 to 10 years, 11 to 12 years, and 13 to 14 years.
Residence was defined as one of eight distinct sectors of Bandiagara town or, in
the case of children from villages nearby Bandiagara, that specific village. Un-
complicated malaria was defined as P. falciparum parasitemia, with an axillary
temperature of ⱖ37.5°C, detected by active surveillance or parasitemia and
symptoms leading to treatment-seeking behavior in the absence of other clear
cause of fever on passive surveillance.
All controls were matched to the index case of severe malaria within 5 days of
initial enrollment. Matched uncomplicated malaria controls were enrolled from
the population of children presenting to the daily Bandiagara Malaria Project
5631
TABLE 1. Defining features of severe malaria (modified) as
published by the World Health Organizationa
Features of severe malaria
Coma (Blantyre coma scale [BCS] ⱕ 2)
Seizure (one or more witnessed by the investigators)b
Obtundation (depressed consciousness with BCS ⬎2)
Parasitemia, ⱖ500,000/mm3
Lethargy or prostration (clinical judgment or child ⱖ7 months
unable to sit unassisted)
Severe anemia (hemoglobin, ⱕ5 g/dl)
Respiratory distress (intercostal muscle retraction, deep breathing,
grunting)
Hypoglycemia (glucose, ⱕ40 mg/dl)
Jaundice
Renal insufficiency as indicated by lack of urination for ⱖ1 day
Gross hematuria
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State of shock (systolic blood pressure, ⱕ50 mm Hg; rapid pulse;
cold extremities)
Inability to eat or drinkc
Repeated vomitingc
a
Definition includes baseline parasitemia and one or more of the listed fea-
tures.
b
Criteria are modified to allow enrollment for children with one witnessed
seizure rather than two to err on the side of safety in this rural setting.
c
Definition included to avoid missing cases of impending severe illness, al-
though these criteria were not used as the sole enrollment criteria.
clinic. Healthy controls were enrolled after traveling to the home of the child
with severe malaria and following a standardized routine of exiting the front
entrance of a compound and making consecutive left turns until another com-
pound with an eligible control was identified. Children were enrolled as healthy
controls if they were asymptomatic for acute illness, had no evidence or history
of chronic illness, and were found to be aparasitemic upon examination.
Study protocols were reviewed and approved by the local Malian Institutional
Review Board, as well as the University of Maryland Institutional Review Board.
Village assent was obtained from village chiefs, government officials, and tradi-
tional healers prior to study initiation. Individual informed consent was obtained
from the legal guardian of each child prior to enrollment, although care for
severe and uncomplicated malaria was offered regardless of study participation.
Clinical processing. At enrollment, clinical information was taken and entered
into standardized forms. Clinical and bedside physical exams were performed
and diagnostic peripheral blood smears, hemoglobin, and glucose were obtained.
An age- and weight-based aliquot of venous blood was obtained for study pur-
poses. Index cases of severe malaria were classified based on modified World
Health Organization criteria and treated for seizure activity, hypoglycemia, se-
vere anemia, or additional complications accordingly. Weight-based intravenous
quinine and intramuscular pyrimethamine-sulfadoxine therapy was used for the
treatment of severe malaria, whereas a standard 3-day course of chloroquine was
used for the treatment of uncomplicated malaria. Sulfadoxine-pyrimethamine
(Fansidar) was used as second-line therapy in the event of treatment resistance
upon follow-up.
Serum collection. Patient whole blood (1 ml) was collected into sterile Ep-
pendorf tubes on admission and prior to institution of antimalarial therapy.
Blood was refrigerated at 4°C and allowed to coagulate for 4 to 6 h prior to
processing via centrifugation. Sera were preserved at ⫺70° C at the field site and
transferred to the University of Maryland at Baltimore for processing by using
liquid nitrogen storage containers. Samples remained frozen until inflammatory
cytokine measurements were performed.
Circulating cytokine and cytokine receptor measurements. Levels of IL-1␤,
IL-6, IL-8, IL-10, IL-12(p70), and TNF-␣ in serum were determined by utilizing
cytometric bead array technology (BD Biosciences, San Diego, Calif.) and flu-
orescence detection by flow cytometry according to the manufacturer’s recom-
mendations with some modifications. This sensitive technique allows the detec-
tion by flow cytometry of multiple cytokines in small quantities of sample. Briefly,
40 ␮l of bead populations with discrete fluorescent intensities of Peridinin chlo-
rophyll protein (PerCP)-Cy5.5 and coated with cytokine-specific capture anti-
bodies were added to 40 ␮l of patient sera, and 40 ␮l of phycoerythrin-conju-
gated anti-human inflammatory cytokine antibodies. Simultaneously, standards
for each cytokine (0 to 5,000 pg/ml) were likewise mixed with cytokine capture
beads and phycoerythrin-conjugated reagent. The vortexed mixtures were al-
5632 LYKE ET AL. INFECT. IMMUN.

TABLE 2. Patient characteristics at enrollment in age, residence-, and ethnicity-matched severe malaria, mild malaria, and healthy
control groups
Severe malaria
Characteristic Uncomplicated malaria Healthy control
Survived Died

No. of subjects 230 18 249 251

Female (%) 121 (53) 10 (56) 124 (50) 117 (47)
Age in mo (range) 41.1 (3–162) 29 (8–99)a 41.5 (6–168) 40.6 (4.5–167)
Hemoglobin concn in g/dla (range) 8.89 (2.6–13.5) 6.12 (2.0–11.8)a 9.5 (5.3–14.2)b 10.6 (6.2–13.4)c
WBC/mm3 13,424 16,124 13,338 11,828c
Parasite densityd 170,029 31,763a 7,820b 0c
a
Denotes significance at the level of P ⬍ 0.05 performed between children that survived and those that died of severe disease. All analyses were performed with
paired Student t test.
b
Paired t test significance (P ⬍ 0.05) determined between children with severe and uncomplicated malaria.
c
Paired t test significance (P ⬍ 0.05) determined between children with uncomplicated malaria and healthy. controls.
d
Data shown are the geometric means. After removal of hyperparasitemic individuals from children with severe disease, the geometric mean parasite density

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remained significantly elevated over children with uncomplicated disease with a mean parasite density of 34,054 asexual parasites/mm3.

lowed to incubate for 3 days, enhancing the lower limit of detection. Flow
cytometric analysis was performed and analyzed by a single operator, and stan-
dard curves were derived from the cytokine standards. The lower limit of detec-
tion for the various cytokines evaluated ranged from 2.5 to 10 pg/ml. For results
above the upper limit of detection, 1:4, 1:8, and 1:16 dilutional experiments were
performed to accurately determine cytokine levels.
Statistical analysis. Pooled analyses of differences in cytokine levels between
clinical groups were performed by using two-sided Student t test for continuous
variables with equal variance (SPSS 10.0; SPSS, Inc., Chicago, Ill.) and Mann-
Whitney rank sum analysis for populations not normally distributed (SigmaStat
3.0; SigmaStat, Chicago, Ill.). To analyze differences in cytokine levels between
matched pairs, the level for statistically significant differences (two-sided) was set
at P ⬍ 0.01.
RESULTS
Patients. Serum for cytokine measurements was available
from 748 of 776 enrolled study children. Table 2 shows the
characteristics at enrollment of these children; the study group
consisted of 248 cases of severe malaria, 249 cases of uncom-
plicated malaria, and 251 healthy controls. The case fatality
rate for severe malaria was 7.3% (18 of 248). Of the children
who died, 7 had cerebral malaria, 5 had combined cerebral
malaria and malaria associated severe anemia, 3 had severe
anemia, and 3 had respiratory distress and/or symptoms of
shock. The mean age of children with severe malaria was 40.5
months, although a significant difference was noted between
those that survived versus those who died (41.4 months versus
29 months; P ⫽ 0.035). Of those with severe malaria, 92 (37%)
were hyperparasitemic (as a sole criterion), 100 (40.3%) had
cerebral malaria, 17 (6.9%) were severely anemic, 18 (7.2%)
had combined cerebral malaria with severe anemia, and 21
(8.5%) met other criteria (i.e., respiratory distress, hypoglyce-
mia, shock, prostration, etc.). Anemia was more common
among those with a fatal outcome than in survivors (mean
hemoglobin concentration of 6.12 g/dl versus 8.89 g/dl, respec-
tively; P ⬍ 0.0001). The mean duration of symptoms until
presentation was 2.8 days in the severe-malaria group.
Differences in cytokine levels between matched groups. Sig-
nificantly elevated levels (shown as geometric mean levels in
picograms/milliliter) of proinflammatory IL-6 (485.2 versus
54.1; P ⬍ 0.001), TNF-␣ (10.1 versus 7.7; P ⬍ 0.001), and
IL-12(p70) (48.9 versus 31.3; P ⫽ 0.004) were noted in severe
cases versus healthy controls (Table 3 and Fig. 1). A borderline
elevation in IL-8 (108 versus 90.2; P ⫽ 0.014) was likewise
noted between these groups. An elevation in anti-inflamma-
tory IL-10 (1,099.3 versus 14.1; P ⬍ 0.001) was seen between
the severe malaria cases and healthy controls. Notably, 218 of
248 (88%) severe cases of malaria had detectable IL-6 levels in
excess of 100 pg/ml, and 227 of 249 (91%) had ⬎200 pg of
IL-10/ml (data not shown). Significantly elevated levels of IL-6
(485.2 versus 141.0; P ⬍ 0.001) and IL-10 (1,099.3 versus 133.9;
P ⬍ 0.001) were noted in severe malaria versus uncomplicated
malaria controls (Fig. 1).
Cytokine levels in study participants with various severe-
malaria manifestations. Subset analysis was performed on pa-
tients presenting with various manifestations of severe malaria

TABLE 3. Inflammatory cytokine results between matched severe P. falciparum malaria, uncomplicated malaria, and healthy, aparasitemic
controls from Bandiagara, Malia
Severe malaria (n ⫽ 248) Uncomplicated malaria (n ⫽ 249) Healthy controls (n ⫽ 251)
Cytokine
b c
Mean pg/ml (range) P Mean pg/ml (range) P Mean pg/ml (range) Pd

IL-1␤ 15.8 (2.5–1,773) 0.283 12.7 (2.5–636) 0.401 14.7 (2.5–3,355) 0.046
IL-6 485.2 (10–33,100) ⬍0.001* 141 (2.6–16,170) ⬍0.001* 54.1 (2.5–11,111) ⬍0.001*
IL-8 108.4 (11.9–19,006) 0.014 107.1 (5.0–29,071) 0.22 90.2 (5.0–19,669) 0.175
IL-10 1,099.3 (10–27,677) ⬍0.001* 133.9 (2.5–23,623) ⬍0.001* 14.2 (3.3–529) ⬍0.001*
IL-12 (p70) 48.9 (2.5–13,304) 0.004* 33.8 (2.5–3,798) 0.026 31.3 (2.5–3,537) 0.439
TNF-␣ 10.1 (2.5–150) ⬍0.001* 8.8 (2.5–1,874) 0.02 7.7 (2.5–2,296) 0.047
a
*, Significant value as determined by Mann-Whitney rank sum analysis with a level of significance set at P ⬍ 0.01.
b
Severe versus healthy.
c
Severe versus uncomplicated.
d
Uncomplicated versus healthy.
VOL. 72, 2004 INFLAMMATORY CYTOKINES IN P. FALCIPARUM MALARIA 5633

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FIG. 1. Distribution of select serum inflammatory cytokine levels in children from Bandiagara, Mali with severe malaria and age-, residency-,
and ethnicity-matched uncomplicated malaria and healthy controls. Geometric mean of cytokine levels (in pg/ml), as well as upper and lower
quartiles, are indicated.

(Table 4 and Fig. 2). Cerebral malaria was associated with malaria cases without cerebral manifestations. Likewise, simi-
significantly elevated levels (shown as geometric mean levels in lar results were found in cases of cerebral malaria combined
picograms/milliliter) of IL-6 (754.5 versus 311.4; P ⬍ 0.001) with severe anemia with elevated levels of IL-6 (797.4 versus
and IL-10 (1,405.6 versus 868.6; P ⫽ 0.006) compared to severe 311.4; P ⬍ 0.001) and IL-10 (1,430.8 versus 868.6; P ⫽ 0.003).

TABLE 4. Subset analysis of cytokine results based on criterion for enrollment as severe malaria

No. of patients Cytokine level (geometric mean pg/ml)c

Severe-malaria Criteriaa
(n ⫽ 248) IL-1␤ IL-6 IL-8 IL-10 IL-12(p70) TNF-␣

Cerebral 100 17.6 754.5 109.5 1,405.6 66.1 10.5

Noncerebral 130b 14.4 311.4 102 868.8 43.6 9.5
P NSc <0.001 NS 0.006 NS NS

Severe anemia 17b 14.7 199.2 84.7 391.1 26.7 9.7

Hemoglobin at ⬎5 g/dl 213 17.2 487.6 106.2 1,160.9 55.2 9.9
P NS 0.03 NS 0.002 NS NS

Cerebral/anemia 18 18.8 797.5 116.2 1,430.8 55.6 10.9

Neither 213 14.4 311.4 102 868.8 43.6 9.5
P NS <0.001 NS 0.003 NS NS

Hyperparasitemia 92 15.4 336.6 116.9 1,007.3 50.8 9.7

⬍500,000/mm3 156 16.1 602.1 103.8 1,157.5 47.8 10.4
P NS 0.002 NS NS NS NS
a
Significance levels were calculated by Mann-Whitney rank sum analysis.
b
Patients with concomitant cerebral malaria and severe anemia were eliminated from analysis.
c
NS, not significant.
5634 LYKE ET AL.
FIG. 2. Distribution of levels of IL-6 and IL-10 in the sera of children with subsets of severe malaria. (a and b) Cytokine levels in children with
or without cerebral malaria; (c and d) cytokine levels in children with hemoglobin (hb) ⬍5 g/dl or ⬎5 g/dl. Geometric mean of cytokine levels (in
pg/ml), as well as upper and lower quartiles, are indicated.
Conversely, significantly lower levels of IL-10 (391.1 versus
1,160.9; P ⫽ 0.002) and borderline differences in IL-6 (199.2
versus 487.6; P ⫽ 0.03) were observed in children with malaria
associated severe anemia compared to severe malaria cases
with hemoglobin at ⬎5 g/dl. Hyperparasitemia was associated
with significantly lower levels of IL-6 (336.6 versus 602.1; P ⫽
0.002). No significant differences were noted in cytokine levels
between children that died (n ⫽ 18) and those who survived
severe malaria (data not shown). Although the results did not
reach statistical significance, a trend in reduced geometric
mean IL-10 was noted in the children that died versus the
children that survived (649.7 versus 1,145.5 pg/ml; P ⫽ 0.16).
The IL-6/IL-10 ratio was not statistically different between
children that died and those that survived (2.12 versus 1.98; P
⫽ 0.96).
Age-specific cytokine levels in volunteers with various man-
ifestations of severe malaria. Age-specific cytokine levels in
children with various manifestations of severe malaria were
analyzed. Comparisons were made between cytokine levels in
children ⱕ24 months old and children older than 24 months in
the severe-malaria group as a whole, as well as by the criteria
for severe disease. No significant differences were noted be-
tween median cytokine levels of any inflammatory cytokine
(data not shown). The small numbers of children older than 72
months limited our ability to stratify children into other age
groups.
INFECT. IMMUN.
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DISCUSSION
Severe P. falciparum malaria is characterized by marked
changes in cytokine production resulting from the immune
response to the infection. Although the body of literature de-
scribing malaria as a systemic inflammatory illness is extensive,
it is also heterogeneous and disperse, with comparisons of
groups often involving few cytokines and small numbers of
subjects of disparate ages, nationalities, and ethnic origins.
Furthermore, the very definition of malaria is complicated with
manifestations varying from mild clinical illness to coma, se-
vere anemia, respiratory distress, and shock. Thus, we sought
to comprehensively examine cytokine production in a large
African cohort of age-, residence-, and ethnicity-matched chil-
dren with severe or uncomplicated malaria and healthy con-
trols and to stratify severe malaria into subsets to determine
whether differences in cytokine levels in serum correlated with
these varied disease manifestations. We found distinct differ-
ences in cytokine production correlating with disease severity
in addition to discrete cytokine patterns in subsets of severe
malaria cases. Our results suggest that cytokine production is a
dynamic process. The limitations inherent in examining cyto-
kine production at a single time point and in circulation rather
than in the local microenvironments complicate the interpre-
tation of these results. Nevertheless, insights could be gained
from the cytokine differences observed between those with
VOL. 72, 2004 INFLAMMATORY CYTOKINES IN P. FALCIPARUM MALARIA
severe malaria and those with uncomplicated malaria and
healthy controls, as well as from distinct cytokine production
patterns that differed between cerebral malaria, severe anemia,
and hyperparasitemia. A discussion of the significance of our
observations follows.
We found significantly elevated levels of proinflammatory
IL-6, IL-12(p70) and, to a lesser extent, TNF-␣ in the sera of
the severe-malaria group compared to age-matched healthy
children. In addition, anti-inflammatory IL-10 was elevated in
the severe-malaria group. IL-6 is an important proinflamma-
tory cytokine that is upregulated by TNF-␣ and acts in concert
with other inflammatory mediators to control parasitemia (18,
48). IL-10 has an important role in immunoregulation, down-
regulating cytokine production (predominantly TNF-␣, IL-6,
and IL-12), inhibiting Th1 function, and promoting natural
killer cell activity (3, 7, 39). Both IL-6 and IL-10 appear to
correlate with disease severity since elevated levels were noted
in the severe-malaria patients compared to the matched un-
complicated malaria cases and, similarly, in uncomplicated ma-
laria cases compared to healthy controls (Table 3). IL-10 ele-
vation also appears to be closely linked to IL-6 production,
supporting the hypothesis that it acts in a counter-regulatory
fashion (9, 21). Previous studies in which serial measurements
of IL-6 and IL-10 were performed revealed an increase in the
IL-6/IL-10 ratio (because of lower IL-10 levels) in samples
drawn prior to death compared to paired survivors (8). Simi-
larly, we noted a relative deficiency in IL-10, albeit not statis-
tically significant, in children that died of severe malaria com-
pared to children that survived, suggesting a loss of
downregulatory function. However, we found no significant
differences in IL-6/IL-10 ratios between these groups due to
the fact that, in contrast to previous data, we found no signif-
icant elevation of IL-6 cytokine levels in children who died
from severe malaria versus those who survived (data not
shown), nor did we find a correlation of age to changes in
cytokine levels. The small number of children that died may
have prevented these findings from reaching statistical signif-
icance.
Although elevations were noted in the geometric mean val-
ues of TNF-␣ and IL-12(p70) in the severe-malaria group,
both the absolute levels (in picograms/milliliter) and the dif-
ferences between enrollment groups were small. Elevated
TNF-␣, produced mainly by blood monocytes and tissue mac-
rophages, has been shown to correlate with disease severity in
multiple studies (5, 8, 18, 19, 48). It is unclear whether the
small differences in cytokine levels in serum noted in our study
are biologically significant. To explore whether the low TNF-␣
levels observed in our subjects were the result of increased
circulating soluble TNF-␣ receptors, we measured soluble
TNF-␣ receptor I (p55/60) or TNF-␣ receptor II (p75/80)
levels in a subset of subjects with various TNF-␣ levels. No
significant correlations were observed between the levels of
soluble TNF-␣ receptors and TNF-␣ (data not shown) in se-
rum, indicating that high levels of TNF-␣ receptors were not
the cause of the observed low levels of TNF-␣ in our study
subjects.
IL-12 plays an important role in the adaptive immune re-
sponse to malaria. Although a correlation with disease severity
has been demonstrated, the levels of IL-12 have been found to
be paradoxically lower in African children with severe malaria
5635
(31, 32, 38), possibly due to inhibition after phagocytosis of
hemozoin or IL-10 induction (36). Although the differences
were small, we found IL-12 to be elevated in cases of severe
malaria, with little significant difference between subsets of
severe malaria. The reasons for the lack of IL-1␤ elevation and
the small TNF-␣ and IL-12 elevations may be the result of
downregulation by IL-10. We hypothesize that an initial rise in
TNF-␣, IL-12, and possibly IL-1␤ resulted in enhanced IL-6
production, followed by increases in IL-10 production as a
counter-regulatory mechanism, leading to blunting of the ini-
tial proinflammatory response. Furthermore, the delay in pre-
sentation for evaluation to clinic (mean, 2.8 days) may have
contributed to the snapshot of cytokine patterns observed at
the time of admission. It is also possible that the cytokine levels
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detected in circulation may not be a true representation of the
levels present in the local microenvironments where the com-
plex cytokine interactions leading to the induction of proin-
flammatory cytokines occurs.
To examine the association of proinflammatory cytokines to
symptoms of severe malaria, children were stratified into those
presenting with cerebral malaria (seizure, obtundation, or
coma), severe anemia, or hyperparasitemia. Although cerebral
malaria is believed to be at one end of the spectrum of severity,
hyperparasitemia, at our site, appears to have less disease
acuity, falling between that of uncomplicated malaria and se-
vere disease. Children presenting with cerebral malaria were
noted to have elevated levels of IL-6 and IL-10 compared to
cases of severe malaria without cerebral manifestations. In
addition, despite published reports of hyperparasitemia corre-
lating with elevated IL-6 levels (8), we found significantly lower
geometric mean levels of IL-6, substantiating our findings that
this is a less acute form of severe malaria at our site and
supporting our assumption that IL-6 correlates with disease
severity.
Malaria-induced anemia is multifactorial, with hemolysis oc-
curring more frequently in nonimmune children and dyseryth-
ropoiesis occurring more often in regions with frequent and
recurrent infections. The role of cytokines in the development
of anemia is not well understood. Murine studies have dem-
onstrated that elevated TNF-␣ levels contribute to bone mar-
row suppression and red cell destruction (4, 46) whereas ele-
vated IL-10 is thought to stimulate hematopoiesis (49). TNF-␣
elevation has been associated with anemia and high-density P.
falciparum infection (45), whereas reduced IL-10 (27), and
IL-10/TNF-␣ ratios have been demonstrated in African chil-
dren with severe malaria-induced anemia (33, 37). After we
stratified the severe malaria cases by enrollment criteria, those
with severe anemia (without the manifestations of cerebral
malaria) were noted to have lower levels of IL-10 compared to
severe malaria cases with hemoglobin at ⬎5 g/dl (P ⫽ 0.001).
In addition, lower levels of IL-6 were noted in the severe-
malaria group, although this observation did not meet our
strict criteria of significance (P ⱕ 0.01). Our result of low IL-10
levels in serum in the absence of significant elevations in
TNF-␣ suggests a potential loss of downregulatory, anti-in-
flammatory function. IL-10 deficiency has been noted in pa-
tients who succumb to malaria (8). We hypothesize that the
severity of disease associated with profound malaria-induced
anemia is the result of a loss of host cytokine regulation. The
absence of significant TNF-␣ elevation suggests that alternate
5636 LYKE ET AL.
counter-regulatory mechanisms aside from the established IL-
10–TNF-␣ relationship may account for these findings.
Very little information is available concerning the role of
IL-8 in the pathogenesis of malaria. IL-8 is a known neutrophil
chemoattractant that has been shown to be elevated in a small
group (n ⫽ 6) of severe, noncerebral P. falciparum adult ma-
laria cases (13). In addition, expression of IL-8 mRNA has
been demonstrated in placental malaria infection (1). We
noted an increase in IL-8 levels that appeared to correlate with
disease severity, although the level of IL-8 did not meet our
strictly defined significance threshold (P ⱕ 0.01). Thus, we
conclude that it is unlikely that IL-8 plays a significant role in
malaria pathogenesis.
The present study is limited by a number of factors. Cytokine
levels have been demonstrated to vary based on circadian
rhythm and within the time course of malaria illness. Longitu-
dinal studies in sporozoite-challenged volunteers have demon-
strated wide variation in the levels of TNF-␣, IL-4, IL-6, and
IFN-␥ in serum (20). Although human immunodeficiency virus
prevalence remains low (⬍2%) in this area, we were unable to
rule out or diagnose concomitant bacterial infection, poten-
tially altering the results. Although clinically apparent pneu-
monia or gastrointestinal infection could be diagnosed at the
bedside, we did not possess culture capability to identify caus-
ative organisms of bacterial infection. Protean infections, such
as nontyphoidal Salmonella spp., appear to correlate with P.
falciparum infection (15, 16) and could have confounded our
results, as could meningitis, the symptoms of which resemble
cerebral malaria. Nevertheless, the majority of children recov-
ered and were discharged after treatment with antimalarial
medication alone, implicating malaria as the sole cause of
symptoms. As mentioned above, it is unclear whether the level
of cytokine detected systemically is reflective of biologically
significant cytokine levels in defined microenvironments (e.g.,
brain, liver, and spleen) or whether a single serum measure-
ment is reflective of a longitudinal process. Thus, additional
studies are necessary to properly assess the impact of the
marked differences in cytokines levels reported in this compre-
hensive analysis in malaria disease severity.
In summary, we have demonstrated in a large cohort of
children that a complex milieu of cytokines is released with
apparent feedback inhibition and cross-regulatory function.
Furthermore, disease states (i.e., cerebral malaria and severe
anemia) within the broadly defined severe-malaria category
have unique and predictable cytokine patterns. Despite these
findings, a deep understanding of the immune response to
malaria remains elusive. Longitudinal studies of malaria dis-
ease states, in conjunction with studies of cytokine production
by peripheral blood mononuclear cells collected from these
children after in vitro incubation with malaria antigens, would
add appreciably to our understanding of the role of cytokines
in disease severity associated with P. falciparum infection.
ACKNOWLEDGMENTS
This study was supported by a contract from the National Institutes
of Allergy and Infectious Diseases (N01-AI-85346).
We thank the Bandiagara Malaria Project staff and the populace of
Bandiagara, Mali, in West Africa, who have been very supportive of
research efforts.
INFECT. IMMUN.
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